Extraction of prostaglandins

ABSTRACT

AN IMPROVEMENT IN A PROCESS FOR ISOLATING PROSTANOIC ACID DERIVATIVES, INCLUDING PGA2, PGA2 METHYL ESTER, PGA2 ACETATE METHYL ESTER, AND THEIR 15B-EPIMERS FROM MARINE INVERTEBRATES. THE PRODUCTS ARE USEFUL FOR PHARMACOLOGICAL PURPOSES AND AS INTERMEDIATES FOR THE PREPERATION OF OTHER PROSTANOIC ACID DERIVATIVES WHICH PHARMACOLOGICAL USES.

United States Patent Oflice 3,794,675 Patented Feb. 26, 1974 US. Cl.260-468 D 14 Claims ABSTRACT OF THE DISCLOSURE An improvement in aprocess for isolating prostanoic acid derivatives, including PGA PGAmethyl ester, PGA acetate methyl ester, and their ISfi-epirners frommarine invertebrates. The products are useful for pharmacologicalpurposes and as intermediates for the preparation of other prostanoicacid derivatives with pharmacological uses.

CROSS-REFERENCE TO RELATED APPLICATIONS This application is acontinuation-in-part of our copending application Ser. No. 71,432, filedSept. 11, 1970, now abandoned.

DESCRIPTION OF THE INVENTION This invention relates to novel methods ofpreparing prostanoic acid derivatives.

This invention relates to a process for producing a compound of theformula:

wherein R and R are both hydrogen, or wherein R is methyl and R ishydrogen or acetyl, which comprises extracting colonies or colony piecesof the marine invertebrate Plexaura homomalla (Esper), 1792, forma S,with a solvent and separating said compound from the resulting extract.

This invention further relates to an improvement in the process forextracting 15,8-PGA acetate methyl ester from colonies or colony piecesof the marine invertebrate Plexaura homomalla (Esper), 1792, forma R, orin a process for extracting PGA acetate methyl ester from colonies orcolony pieces of the marine invertebrate Plexaura homomalla (Esper),1792, forma S, wherein said colonies or colony pieces are cooled to atemperature at least as low as +5 C. within one hour after removal ofsaid colonies from their growing site, said colonies or colony piecesbeing maintained at a temperature at least as low as +5 C. until thetime they are extracted with a neutral organic liquid.

This invention further relates to an improvement in the process forextracting prostanoic acid derivatives from colonies or colony pieces ofthe marine invertebrate Plexaura homomalla (Esper), 17'92, forma S orthe marine invertebrate Plexaura homomalla (Esper), 1792, forma R,wherein said colonies or colony pieces, prior to extraction, aremaintained in contact with water in a temperature range up to 50 C.until substantially all of the Cl5 acetates of the prostanoic acidderivatives originally present in the colonies or colony pieces aretransformed to C-15 hydroxy prostanoic acid derivatives.

In particular, the several aspects of this invention relate toderivatives of prostanoic acid which has the following structure andnumbering:

Some of the derivatives of prostanoic acid are known as prostaglandins.One of those, prostaglandin E (PGE has the following formula:

II Another, prostaglandin F (PGF has the formula:

coon

Still another, prostaglandin F (PGF has the formula: A

Encompa-ssed by Formula I are the prostanoic acid derivatives known asprostaglandin A and its esters, namely PGA (R and R are hydrogen), PGA'methyl ester (R is methyl and R is hydrogen), and PGA acetate methylester (R is methyl and R is acetyl). These are known compounds, known tobe useful for pharmacological purposes. See, for example, Britishspecification No. 1,097,533 and Bergstrom et al., Pharmacol. Rev. 20, l(1968). These Formula-I compounds are also useful as intermediates forthe preparation of other prostanoic acid derivatives withpharmacological uses.

In Formulas I to IV and in the formulas recited hereinafter in thespecification and claims, broken line attachments to the cyclopentanering indicate substituents in alpha configuration, i.e., below the planeof the cyclopentane ring. Heavy solid line attachments to thecyclopentane ring indicate substituents in beta configuration, i.e.,above the plane of the cyclopentane ring.

The side-chain hydroxy at 0-15 in Formulas I to IV is in S (alpha)configuration. That configuration is shown by attachment of saidside-chain hydroxy to C-15 with a dotted line and hydrogen with a heavysolid line. The alternative configuration for the side-chain hydroxy atC-lS is known as R or epi (beta), and is shown when necessary byattachment of said side-chain hydroxy to C-15 with a heavy solid lineand hydrogen with a dotted line, thus H on The prostaglandincorresponding to PGE (Formula H) but with the R or epi configuration atC-IS will be des- 3 ignated fi-PGB See Nature, 212, 38 (1966) fordiscussion of the stereochemistry of the prostaglandins.

These conventions regarding formulas, names, and symbols for derivativesof prostanoic acid apply to the formulas, names, and symbols givenhereinafter in the specification and claims. When reference is madeherein after to the compounds of Formulas I to IV, by the symbos PGA PGEPGF or PGF or to the methyl esters of any of those, 15(8) configurationwill be intended and by established custom, S will not be mentioned inthe name or symbol. For all of the other compounds recited hereinafter,the configuration at C-lS will be identified in the name as 15,8 when inthe 15 (R) or 15-epi configuration.

Molecules of the known prostaglandins each have several centers ofasymmetry, and can exist in racemic (optically inactive) form and ineither of the two enantiomeric (optically active) forms, i.e., thedextrorotatory and levorotatory forms. As drawn, Formulas I to IV eachrepresent the particular optically active form of the prostaglandinwhich is obtained from certain mammalian tissues, for example, sheepvesicular glands, swine lung, or human seminal plasma, or by carbonyland/ or double bond reduction of a prostaglandin so obtained. See, forexample, Bergstrom et al., Pharmacol. Rev. 20, 1 (1968) and referencescited therein.

The several aspects of this invention, then, relate to novel andimproved methods of extracting and separating PGA PGA methyl ester, andPGA acetate methyl ester from Plexaura homomalla (Esper), 1792, forma S,and 15fiPGA 155-PGA methyl ester, and 15B-PGA acetate methyl ester fromPlexaura homomalla (Esper), 1792, forma R. These procedures will bediscussed below and illustrated in the examples. Also discussed below,together with Charts A-E, and illustrated in the preparations will bethe transformations of these PGA and 1-55- PGA compounds to otherprostaglandins, particularly PGE PGFg and PGF These transformations arenot part of the present invention but are illustrative of the utility ofthis invention.

The known prostanoic acid derivatives, PGE PGF and PGF- and their estersand pharmacologically acceptable salts, are extremely potent in causingvarious biological responses. For that reason, these compounds areuseful for pharmacological purposes. See, for example, Bergstrom et al.,Pharmacol. Rev. 20, 1 (1968), and references cited therein. -A few ofthose biological responses are vaso-depressor activity in the case ofthe PGE PGF and PGA- compounds as measured, for example, in anesthetized(pentobarbital sodium) pentolinium-treated rats with indwelling aorticand right heart cannulas; pressor activity, similarly measured, for thePGF compounds; stimulation of smooth muscle as shown, for example, bytests on strips of guinea pig ileum, rabbit duodenum, or gerbil colon;potentiation of other smooth muscle stimulants; antilipolytic activityas shown by antagonism of epinephrine-induced mobilization of free fattyacids or inhibition of the spontaneous release of glycerol from isolatedrat fat pads; inhibition of gastric secretion 'in the case of the PGEand PGA compounds as shown in dogs with secretion stimulated by food orhistamine infusion; activity on the central nervous system; controllingspasm and facilitating breathing in asthmatic conditions; decrease ofblood platelet adhesiveness as shown by platelet-to-glass adhesiveness,and inhibition of blood platelet aggregation and thrombus formationinduced by various physical stimuli, e.g., arterial injury, and variousbiochemical stimuli, e.g., ADP, ATP, serotonin, thrombin, and collagen;and in the case of the PGE compounds, stimulation of epidermalproliferation and keratinization as shown when applied in culture toembryonic chick and rat skin segments.

Because of these biological responses, these known prostaglandins areuseful to study, prevent, control, or

alleviate a wide variety of diseases and undesirable physioligicalconditions in birds and mammals, including humans, useful domesticanimals, pets, and zoological specimens, and in laboratory animals, forexample, mice, rats, rabbits, and monkeys. With particular regard to PGEsee, for example, Karim et al., Br. Med. J. 3, 198-200 1970), and J.Obstet. Gynaec. Br. Commonw. 77, 200- 210 (1970); as to PGFF see, forexample, Wigvist et al. The Lancet 889 (1970), and Karim et al., Lancet1, 157- 159 (1970) and J. Obstet. Gynaec. Brit. Commonw. 76, 769 (1969);as to PGA see, for example, British specification 1,097,533.

For example, these compounds, especially the PGE compounds, are usefulin mammals, including man, as nasal decongestants; the PGE and PGAcompounds are useful to reduce and control excessive gastric secretion,thereby reducing or avoiding gastrointestinal ulcer formation, andaccelerating the healing of such ulcers already present in thegastrointestinal tract; the PGE compounds are extremely potent incausing stimulation of smooth muscle; the PGE and PGF compounds areuseful as hyptotensive agents to reduce blood pressure in mammals,including man; the PGA compounds increase the flow of blood in themammalian kidney, thereby increasing volume and electrolyte content ofthe urine; the PGE PGF and PGF compounds are useful for labor inductionand abortion in pregnant female animals, including humans. For thesepurposes, these compounds are administered by the usual intravenous,intramuscular, or subcutaneous injection or infusion methods.

The various PGE PGF PGF2B, and PGA acids are readily converted to estersor pharmacologically acceptable salts, for purposes of administration,by methods known in the art.

As stated above, the Formula I PGA compounds are known in the art.Considering the corresponding 15 3- PGA compounds, both 15flPGA acetatemethyl ester and 15fiPGA are known in the art. See, for exampleWeinheimer et al., Tetrahedron Letters, No. 59, 5185 (1969); and H. W.Youngken, Jr. (ed.), Food-Drugs From the Sea, Proc. Marine TechnologySociety, pp. 311-314 (1969).

The compounds of Formula I and the two -PGA compounds above are obtainedby extraction from tWo forms of a marine invertebrate. The Formula I PGAcompounds are obtained from colonies of Plexaura h0- momalla (Esper),1792, forma S; the 15 3-PGA compounds are obtained from colonies ofPlexaura homomalla (Esper), 1792, forma R. It has been reported that15fiPGA and 15flPGA acetate methyl ester are present in the air-driedcortex of Plexaura h momalla to the extent of 0.2% and 1.3%,respectively. See Weinheimer et al. cited above. I

The Plexaura homomalla forms are members of the subclass Octocoorallia,order Gorgonacea, suborder Holaxonia, family Plexauridae, genusPlexaura. See, for example, Bayer, The Shallow-Water Octocorallia of theWest Indian Region, Martinus Nijhoff, The Hague (1961). Colonies ofthese Plexaura homomalla forms are abundant on the ocean reefs in thezone from the lowtide line to about 25 fathoms in the tropical andsubtropical regions of the western part of the Atlantic Ocean, fromBermuda to the reefs of Brazil, including the eastern shore reefs ofFlorida, the Caribbean island and mainland reefs, and the Gulf of Mexicoisland and mainland reefs. These colonies are bush-like or smalltree-like in habit, and are readily identified for collection asPlexaura homomalla (Esper), 1792, by those of ordinary skill in thisart. One method of distinguishing the S form from the R form isdescribed below in Preparation 1.

The colonies of these two forms of Plexaura homomalla are easilyseparated into an outer bark-like cortex and an inner wiry proteinaceousstem or skeleton. Symbiotic algae or Zooxanthellae are also present inthe colonies.

The choice of isolation or extraction method is determined by theparticular PGA (Formula I) or 1513- PGA type compound desired. Thus ofPGA acetate methyl ester, PGA methyl ester, and PGA are desired, freshlycollected or air-dried S-form colonies or colony pieces are extracted;for maximum convenience and maximum yield of either PGA acetate methylester or 15/3-PGA acetate methyl ester, the respective S or R- formcolonies or colony pieces, freshly collected, are frozen prior toextraction. If mainly PGA or 15/i-PGA is desired, the respective S orR-form colonies or colony pieces are maintained in contact with wateruntil free of C-15 acetates, with optional freezing before or aftermaintaining in contact with water.

Prior to extracting the Formula I products, the freshly collectedcolonies or colony pieces are advantageously broken or chopped intosmaller pieces, preferably less than 2 grams in weight, prior toextraction. For some extraction systems it is desirable to subdivide thecolony pieces to give a surface area increase in the range 100 to100,000 times the surface area of an equal weight of colonies.

Alternatively, the colonies or colony pieces are airdried beforeextraction. When that is done, it is advantageous to separate the thickbark-like cortex from the wiry proteinaceous skeleton, and thenpulverize the cortex before extraction. The major amount of the FormulaI compounds is in said cortex.

The extraction solvents are any of the usual neutral organic liquidsused for extraction purposes, for example hydrocarbon, halogenatedhydrocarbons, lower alkanols, ketones and esters. Preferably, thesolvent has a boiling point below about 100 C. Examples of preferredsolvents are benzene, dichloromethane, methanol, ethanol, acetone, andethyl acetate.

Separation of the Formula I products from the extract is done by methodsknown in the art, for example liquidliquid extraction or silica gelchromatography.

Since the invention of the novel and improved processes for producingthe Formula I compounds and their 15B-epimers, it has now been foundthat small amounts of the 5,6-trans compounds of PGA and 15,8-PGA andtheir methyl esters and acetate methyl esters are also ob tained fromPlexaura homomalla (Esper), 1792, forms R and S. These 5,6-transcompounds are extracted with and accompany the corresponding PGA -typecompounds through many of their transformations. For example, PGAcontaining 5,6-trans-PGA yields a mixture of PGE and 5,6-trans-PGE bythe process represented in Chart E below.

When it is desired, for pharmacological purposes, to prepare the majorproducts of this invention free of 5,6- trans compounds, those 5,6-transcompounds are separated either from the starting reactants or from theproducts. In either case, several methods are available for separatingthe 5,6-trans-PG compounds from the PG; compounds. One method is bymeans of a silver-saturated ion-exchange resin (for example, see E. A.Emken et al., I. Am. Oil Chemists Soc. 41, 388 (1964)), illustratedbelow in Preparation 2. The other method is by preferentially forming amercuric acetate adduct of the 5,6-cis compound which is extractableinto polar solvents illustrated below in Preparation 3. Accordingly, theremoval of 5,6-trans-PGA acetate methyl ester from PGA acetate ester isdone advantageously by the procedure of Preparation 3 below.

For maximum yield of either PGA acetate methyl ester or 15fl-PGA acetatemethyl ester, there is now an improvement in the process of extractingcolonies or coloriy pieces of either Plexaura hamomalla (Esper), 1792,forma S or Plexaura homomalla (Esper), 1792, forma R, respectively.

In this improved process, said colonies or colony pieces are cooled to atemperature at least as low as +5 C. within about one hour after removalof said colonies from their growing site, said colonies or colony piecesbeing maintained at a temperature at least as low as +5 Cruntil the timethey are extracted with a neutral organic liquid.

When these colonies or colony pieces are treated in this manner, asurprising and unexpected difference is observed in the composition ofthe prostanoic acid derivative mixture which is extracted, compared withthe prior art. For example, Weinheimer et al., cited above, disclose theisolation of one part of 15,8-PGA and 6.5 parts of 15,8-PGA acetatemethyl ester from the air-dried cortex of Plexaura homomalla colonies.By using the novel improvement of this invention, a substantiallysmaller ratio of hydroxy compound to acetate compound is obtained. Forexample, in Example 3, below, a ratio of about 1 part of hydroxycompound to about 25 parts of acetate compound is obtained. Thisunexpected result is attained using the novel improvement of thisinvention with colonies 01' colony pieces of both Plexaura homomalla(Esper), 1792, forma R and Plexaura homomalla (Esper), 1792, forma S.This result is desirable when the acetate compound is desired as anintermediate.

A temperature below about +5 C. is readily obtained by a mixture of iceand water, and it is convenient to immerse the colonies or colony piecesin that mixture as soon as convenientafter the colonies are removed fromtheir growing site. However, in a preferred embodiment of this processimprovement, all or part of the cooling is done at a temperature belowabout 20 C. Thus, the colonies or colony pieces are actually frozen andkept frozen until the time for extraction. Preferably, the colonies orcolony pieces are frozen to below about 20 C. within about one hourafter removal from the growing site, and are maintained below about 20C. until the time for extraction.

In another preferred embodiment of this process improvement, thecolonies or colony pieces are frozen by contact with solid carbondioxide (Dry Ice) or are placed in a container which contains solidcarbon dioxide, the resulting frozen colonies or colony pieces beingmaintained in contact with said carbon dioxide or at an equivalenttemperature until the time for extraction.

The frozen colonies or colony pieces are advantageously broken orchopped into smaller pieces, preferably less than 2 grams in weight,prior to extraction. For some extraction systems it is desirable togrind the colony pieces in a lhogger to a particle size with the largestdimension about 5 mm. The degree of subdivision is selected according toseveral factors, for example, the manner in which the solids arecontacted by the extracting liquid, whether by percolation through astatic bed or by agitation and stirring, the contacting time, the natureand viscosity of the solvent, and the like.

The neutral organic liquid used to extract the colonies or colony piecesis any of the usual organic solvents, preferably one with a boilingpoint below about C. and with moderate to high polarity. Especiallypreferred liquids for this extraction are dichloromethane, methanol, andethanol. Unnecessarily prolonged contact of the colonies or colonypieces with a hydroxylic organic solvent, e.g., methanol or ethanol,should, however, be avoided since some of the desired acetate may betransformed in the solvent to the corresponding hydroxy compound. Anoptimum duration of extraction is readily determined by those ofordinary skill in this art, for example by repeated extraction of asmall amount of colony pieces with fresh portions of the solvent todetermine how much time is required for substantially completeextraction. The extraction temperature is not critical as long as theextraction is not done at a temperature so high that the desiredproducts are decomposed. Extraction in the temperature range 5 to 30 C.is usually satisfactory.

The prostanoic acid derivatives are isolated from the extract and areseparated from each other by procedures known in the art. Thoseprocedures are exemplified below in the examples.

For maximum yield of either PGA or 15fi-PGA there is now an improvementin the process of extracting colonies or colony pieces of eitherPlexaura homomalla (Esper), 1792, forma S or Plexaura homomalla (Esper),1792, forma R, respectively. In this improved process said colonies orcolony pieces, prior to extraction, are maintained in contact with waterin a temperature range up to 50 C. until substantially all of the C-15acetates of the prostanoic acid derivatives originally present in thecolonies or colony pieces are transformed to C-15 hydroxy prostanoicacid derivatives.

The water referred to above may be cell water, i.e. water within theinternal structure of the marine invertebrate, water within the endodermstructure and the spicules, water adhering to the colonies as they arecollected, or water added for the purpose of increasing the yield ofPGA; or ISB-PGA With regard to the natural internal water of these formsof Plexaura homomalla (Esper), 1792, about one-half of a colony weightconsists of internal water as shown by heating both the cortex or outerbark and the wiry proteinaceous skeleton separately at 50 C. and 0.2 mm.pressure for 24 hours, and then at 50 C. and atmospheric pressure for anadditional 48 hours. The quantity of water present during the periodwherein all of the C-15 acetates are transformed to C-15 hydroxyderivatives is at least equal in weight to the dry solids content of thecolonies or colony pieces. To prevent loss of the water by evaporation,the colonies or colony pieces are either enclosed in containers orsubmerged.

In one embodiment of this process improvement, for convenience inbreaking the colonies to smaller pieces, the colonies or colony piecesare frozen prior to chopping or grinding. This is done by methods knownin the art.

For some reason not fully understood, superior results as to yield ofPGA or ISfl-PGA are obtained if, after the colonies or colony pieces aremaintained in water until the C-l5 acetates are transformed to C-hydroxy derivatives, said colonies or colony pieces are frozen andthawed prior to extraction. In their frozen condition the colonies orcolony pieces may be stored conveniently without gross deterioration orundesirable chemical transformations and side-reactions.

During their period of transformation of Cl5 acetates to C-15 hydroxyderivatives, the mixture of colonies or colony pieces in contact withwater, some of which may be added water, are maintained in the range to40 C. until the desired chemical change takes place. At highertemperatures, i.e., above about 50 C., the chemical change takes placemore rapidly, but there is a possibility that some degradation of thedesired product takes place. At lower temperatures, i.e., down to about5 C., the desired chemical change takes place more slowly. At about C.,the desired reaction is substantially complete within about 15 to 20hours.

Completion of the desired reaction, i.e., transformation ofsubstantially all of the C-15 acetates of the prostanoic acidderivatives originally present in the colonies or colony pieces to C-15hydroxy prostanoic acid deriva tives is readily determined by removing acolony piece or a few small pieces and extracting those with methanol.This methanol extract is subjected to thin layer chromatography with theAIX system. Eventual substantial absence of the 15-acetates is readilyobserved.

As mentioned above, the principal prostanoic acid derivatives in thesetwo forms of Plexaura homomalla are PGA acetate methyl ester and15-epi-PGA acetate methyl ester. The process improvement described abovechanges substantially all of the acetate moieties to hydroxy moieties.At the same time, most of the carboxymethyl moieties, i.e., --COOCH arechanged to carboxyl moieties, i.e., -COOH. Usually, however, at the timesubstantially all of the acetate moieties have been changed to hydroxymoieties, a small amount of IS-hydroxy methyl esters is still present.If desired, the mixture of water and thawed colonies or colony piecescan be maintained in the range up to 50 C. until those 15-hydroxy methylesters are transformed to 15-hydroxy acids. But the amounts ofIS-hydroxy methyl esters remaining are usually small, and the esters areeasily separated from the 15-hydroxy acids. Therefore it is optional inthis process improvement whether one continues maintaining the thawedcolonies or colony pieces beyond the point where LS-acetate compoundsare substantially absent.

When the desired transformation of 15-acetate compounds to IS-hydroxycompounds is substantially complete, the hydroxy acids and hydroxymethyl esters are isolated from the colonies and colony pieces and fromthe water which is also present by suitable extraction techniques. Forthis purpose it is preferred that a water-miscible organic liquid beused, for example, a lower alkanol such as methanol or ethanol, or aketone such as acetone, preferably with a boiling point below about C.

The solution of the extract and organic liquid is concentrated,preferably at temperatures below about 50 C., as with reduced pressure.If foaming is troublesome, an antifoam agent is added, for example apolypropylene glycol, lard oil, soybean oil, a silicone, or othermaterial useful for this purpose as known in the art.

As with the other Formula I compounds and their 1513- epimers above, theseparation of PGA or ISB-PGA from the extract is done by methods knownin the art, for example silica gel chromatography. Likewise, thecorresponding 5,6-trans compounds are separated by the methods disclosedabove, for example using a silver resin column.

An alternate route to PGA or 15B-PGA is from the corresponding methylesters and 15-acetate methyl esters after those have been extracted fromthe Plexaura homomalla colonies or colony pieces as described above. Asuitable method for removing the acetyl group of the Formula I1S-acetate methyl ester of its 15e-epimer comprises mixing the acetatemethyl ester in lower alkanol solution, preferably in methanol solution,with a strong acid, e.g., perchloric acid, for about 15 hours at 25 C. Asuitable method for removing the methyl group of the Formula I methylester or its 15 fi-epimer is the enzymatic hydrolysis described in WestGer-many Otfenlegungschrift No. 1,937,912, reprinted in Farmdoc CompleteSpecifications, Book No. 14, No. 6869R, Week R Mar. 18, 1970.

Transformations of the above PGA and 15fi-PGA compounds to otherprostaglandins, e.g. PGE PGF and PGF or their 15,8-epimers are discussedbelow, but are not part of the present invention.

When PGE-type compounds are desired, the PGA-type compounds of Formula Iand their 15p-epimers are transformed by the chemical reactions showngenerically in Chart A.

In Chart A, R is hydrogen, methyl, or Si(A) wherein A is alkyl of one to4 carbon atoms, inclusive, aralkyl of 7 to 12 carbon atoms, inclusive,phenyl, or phenyl substituted with one or 2 fiuoro, chloro, or alkyl ofone to 4 carbon atoms, inclusive; G is H OH;

H om

wherein R is hydrogen, acetyl, or Si(A) when R is hydrogen or methyl,and R is Si-(A) when R is --Si(A) R is hydrogen or methyl; and B is H OH9 CHART A VII l (hydrolysis) VWV g l B HO VIII Thus, Formula V in ChartA encompasses the compounds of Formula I and their ISB-epimers obtainedfrom Plexaura homomalla, and also compounds of the formula:

O-Si(A) 11 Si-(A);

wherein A is as defined above.

In Formula VI, of Chart A,

ONW

indicates attachment of the epoxy oxygen to the ring in alpha or betaconfiguration. In Formulas VII and VIII of Chart A, indicates attachmentof hydroxy to the ring in alpha or beta configuration.

It will be observed in Chart A that the Formula VII and VIII productseach encompass four stereoisomeric groups of compounds. Included arecompounds with the 11a,15 (S) configuration of PGE (Formula II, above),and compounds with the configuration of 11a,15 8-PGE as obtained fromPlexaura homomalla (Esper), 1792, forma R. If the Formula VII or VIIIproduct is to have the 15 (S) configuration, e.g., PGE then the FormulaV 16 starting material should have the 15 (S) configuration, i.e., Gshould be If a 15 3 compound of Formula VII or VIII is desired, e.g.,15fi-PGE then the Formula V starting material should have the 15pconfiguration, i.e., G should be As described above, Formula V startingmaterials wherein R is hydrogen or methyl and G is i.e. with the 15(8)configuration, are obtained from Plexaura homomalla (Esper), 1792, formaS. Those same compounds are also produced by reacting the corresponding15;? compound with a hydrocarbyl or halohydrocarbyl sulfonyl chloride orbromide, preferably a lower alkylsulfonyl chloride or bromide,especially methanesulfonyl chloride or bromide, or a benzeneorsubstituted-benzenesulfonyl chloride or bromide, e.g., p-toluenesulfonylchloride. This reaction is done in the presence of at least sufiicienttertiary amine, e.g., triethylamine, to absorb the hydrogen chloride orhydrogen bromide byproduct, and at a low temperature, preferably in therange 15 to +15 C. The presence of an inert liquid diluent, e.g.,tetrahydrofuran, is helpful to maintain a mobile homogeneous reactionmixture. At 0 C. and with methanesulfonyl chloride, usually 30 to 60minutes is a sufiicient reaction time. The product is a mixture of 15(S)(alpha) product and 15(R) (beta) starting material. These are separatedby procedures known in the art, and the 15(S) product is purified byprocedures known in the art, advantageously by chromatography on silicagel. This reaction is also used to transform 15 (S) Formula I materialswherein R is hydrogen or methyl and R is hydrogen to the corresponding15(R) compounds. In each case, a mixture of 15(R) product and 15(5)starting material is obtained, the components of which are separated asdescribed above.

Referring again to Chart A, the transformation of starting material V toepoxide VI is carried out by reacing V with any agent known to epoxidizean ate-unsaturated ketone without reacting with isolated carbon-carbondouble bonds, for example see Steroid Reactions, Carl Djerassi, ed.,Holden-Day Inc., 1963, p. 593. Especially preferred are aqueous hydrogenperoxide or an organic tertiary hydroperoxide. See, for example, OrganicPeroxides, A. V. Tobolsky et al., Interscience Publishers, New York,1954. For this purpose, the peroxide or hydroperoxide is employed in anamount of at least one equivalent per mole of Formula V reactant in thepresence of a strong base e.g., an alkali metal hydroxide, a metalalkoxide, or a quaternary ammonium hydroxide. For example, there isemployed lithium hydroxide, sodium hydroxide, potassium hydroxide,lithium ethoxide, lithium octyloxide, magnesium rnethoxide, magnesiumisopropoxide, benzyltrimethylammonium hydroxide, tetraethylammoniumhydroxide, butyltrimethylammonium hydroxide, butyldiethylphenylammoniumhydroxide, benzylethyldimethylammonium hydroxide,benzyldimethyloctadecylammonium hydroxide, benzyldodecyldimethylammoniumhydroxide, decyldimethylphenylammonium hydroxide, and the like. See, forexample, Sidgwick, Organic Chemistry of Nitrogen, third edition, rev. byMiller and Springall, Oxford, 1966, pp. 116-127.

It is advantageous to use an intert liquid diluent in the epoxidationstep to produce a mobile homogenous reaction mixture, for example, alower alkanol, dioxane, tetrahydrofuran, dimethoxyethane,dimethylsulfoxide, or dimethylsulfone. When the alpha epoxide ispreferred, tetrahydrofuran or the less polar dimethoxyethane areespecially preferred as the diluent. A reaction temperature in the range60" to C. is generally preferred, especially below 10 C. The lowertemperatures below 30 C. are especially preferred for favoring formationof alpha epoxide over beta epoxide. At a temperature of 20 C. theepoxidation is usually complete in 3 to 6 hours. It is also preferredthat the reaction be carried out in an atmosphere of an inert gas, e.g.,nitrogen, helium, or argon. When the reaction is complete as shown bythe absence of starting material on TLC plates (3% acetone indichloromethane), the reaction mixture is neutralized, and the epoxyproduct is isolated by procedures known in the art, for example,evaporation of the diluent and extraction of the residue with anappropriate water-immiscible solvent, e.g., ethyl acetate.

This transformation of V to VI usually produces a mixture of Formula VIalpha and beta epoxides both with either the 15 (R) or 15 (S)configuration depending on the configuration at C-lS in the Formula Vstarting material. Although these mixtures are separated into theindividual alpha and beta isomers, for example, by chromatography byprocedures known to be useful for separating alpha and beta epoxidemixtures, it is usually advantageous to transform the Formula VI mixtureof alpha and beta epoxides to the corresponding mixture of Formula VII,Hot and H hydroxy compounds. The latter mixture is then readilyseparated into the Hot and 11,8 compounds, for example, bychromatography of silica gel.

As mentioned above, the starting materials of Formula V encompass notonly the Formula I compounds and their ISB-epimers obtained fromPlexaura homomalla but also the silyl compounds of Formula Va. Whendesired as reactants, these silyl compounds are prepared by silylationof PGA ISp-PGA or the methyl esters of those. These silylations arecarried out by procedures known in the art. See, for example, Pierce,Silylation of Organic Compounds, Pierce Chemical Co., Rockford, Ill.(1968). The Cl5 hydroxy group of PGA 15fi-PGA or their methyl esters istransformed to a OSi(A) moiety wherein A is as defined above, suflicientsilylating agent being used according to known procedures to accomplishthat. The necessary silylating agents for this purpose are known in theart or are prepared by methods known in the art. See, for example, Post,Silicones and Other Organic Silicon Compounds, Reinhold PublishingCorp., New York, NY. (1949). In the case of PGA and 15/3- PGAg, excesssilylating agent and prolonged treatment also transform the COOH toCOOSi(A) It is optional in transforming V to VI whether or not this COOHof PGA or 15/3-PGA is esterified to CO OSi (A) since that ester group istransformed to COOH during formation and isolation of the Formula VIepoxide product.

The various As of a Si(A) are alike or different. For example, a Si-(A)can be trirnethylsilyl, dimethylphenylsilyl, or methylphenylbenzylsilyl.

When it is desired to retain the Si--(A) moiety at C-15 in the FormulaVI epoxide product, for example, to give steric control in a subsequentreaction, it is important in isolating the epoxide that the presence ofacid be avoided and that contact with water be minimized unless thewater is kept cold, i.e., below about 10 C.

Referring again to Chart A, the transformation of epoxide VI to hydroxycompound VII is accomplished by reduction with chromium (II) salts,e.g., chromium (II) chloride or chromium (II) acetate. Those salts areprepared by methods known in the art, e.g., Inorganic Syntheses, VIII,125 (1966); ibid. VI, 144 (1960); ibid. III, 148 (1950); ibid. I, 122(1939); and references cited in those. This reduction is carried out byprocedures known in the art for using chromium (II) salts to reduceepoxides of nip-unsaturated ketones to {S-hydroxy ketones. See, for

12 example, Cole et al., J. Org. Chem. 19, 131 (1954), and Neher et al.,Helv. Chem. Acta 42, 132 (1959). In these reactions, the absence of airand strong acids is desirable. If it is desired to maintain a Si-(A)moiety on C-l5, a neutral reaction mixture is preferred. An especiallypreferred procedure is to generate the chromium (II) ion in the presenceof the Formula VI epoxide, for example, by mixing the epoxide with achromium (III) salt, e.g., the chloride, with metallic zinc in thepresence of acetic acid. The desired Formula VII compound is isolatedfrom the reduction reaction mixture by methods known in the art,

care being taken to minimize contact of the product with acid and water,especially warm water, when retention of a Si-(A) at 6-15 is desired.

Unexpectedly, amalgamated aluminum metal has also been found to beuseful as a reducing agent in place of chromium (II) salts to transformFormula VI epoxides to Formula VII hydroxy compounds. Amalgamatedaluminum is prepared by procedures known in the art, for example, bycontacting aluminum metal in the form of foil, thin sheet, turnings, orgranules with a mercury (II) salt, for example, mercuric chloride,advantageously in the presence of sufficient water to dissolve themercury (II) salt. Preferably, the surface of the aluminum metal is freeof oxide. That is readily accomplished by physical removal of the usualoxide layer, e.g., by abra sion or scraping, or chemically, e.g., byetching with aqueous sodium hydroxide solution. It is only necessarythat the aluminum surface be amalgamated. The amalgamated aluminumshould be freshly prepared, and maintained in the absence of air andmoisture until used.

The reductive opening of the Formula VI epoxide ring is accomplished bycontacting said epoxide with the amalgamated aluminum in the presence ofa hydroxylic solvent and sufficient inert organic liquid diluent to givea mobile and homogeneous reaction mixture with respect to the hydroxylicsolvent and said epoxide. Among hydroxylic solvents, water is especiallypreferred although lower alkanols, e.g., methanol and ethanols are alsooperable.

Examples of inert organic liquid diluents are normally liquid etherssuch as diethyl ether, tetrahydrofuran, dimethoxyethane, diglyme(dimethyl ether of diethylene glycol), and the like. Especiallypreferred is tetrahydrofuran. When a water-immiscible liquid diluent isused, a mixture of water and methanol or ethanol is especially useful inthis reaction since the latter two solvents also aid in forming thedesired homogeneous reaction mixture. For example, a mixture of diethylether and water is used with sutlicient methanol to give a homogeneousreaction mixture.

The reductive opening of the epoxide is carried out by mixing a solutionof the epoxide in the organic diluent with the amalgamated aluminum andthe hydroxylic solvent. Since the reaction is exothermic, it is usuallyadvantageous to cool the solution to a low temperature, e.g., minus 20C. to 0 C., before adding the amalgamated aluminum and hydroxylicsolvent and to maintain the reaction mixture in the range 20 to 30 C. byexternal cooling. This is especially advantageous when water is used asthe hydroxylic solvent. Higher reaction temperatures are operable butnot preferred when a high yield of the Formula VII products is desired.Stirring is preferred during the reaction since the reaction mixture isheterogeneous with respect to the solution and the amalgamated aluminum.

The course of the reaction is advantageously determined by withdrawingsmall portions of the solution and determining the presence or absenceof starting material by thin layer chromatography. For example, when Ris methyl and G is H O COCH;

in, Formulas VI and VII, a suitable TLC system is ethylacetate-cyclohexane-acetic acid (40/60/2), the Formula VI startingmaterial having r, 0.64, and the two Formula VII products having r 0.25II/3) and r; 0.20 (Ila).

The desired Formula XII hydroxy products are isolated by filtration ofthe reaction mixture, advantageously after addition of magnesium sulfateas a filter aid, and evaporation of the organic diluents. The FormulaVII products are then hydrolyzed if desired to remove Si-(A) from -15,and the Ho: and 115 products of Formula VIII are separated, if desired,by procedures known in the art, e.g., chromatography on silica gel.

The products of Formula VII are all of the PGE -type and include PGE PGE15-acetate, PGE methyl ester, PGE IS-acetate methyl ester, PGE and PGEmethyl ester with a -OSi-(A) at C-15, the corresponding 15;? compounds,and compounds corresponding to all of those wherein hydroxy is attachedto C-ll in beta configuration.

Still further regarding the Formula VII and VIII compounds, either asmixture or separately, any of those is transformed to other usefulcompounds or mixtures by changing these PGE-type compounds to PGF-typeproducts by reducing the ring carbonyl at C-9 to alpha hydroxy or betahydroxy. Those transformations are shown in Chart B.

In'Chart B, R is hydrogen, methyl, or Si-(A)a, R is hydrogen or methyl,R is hydrogen or Si(A) and G is H CH4 wherein R is hydrogen or -Si(A)when R and R are hydrogen; R is hydrogen, acetyl, or Si(A) when R and Rare methyl; and R is Si(A) when R is 4i--(A) wherein A is as definedabove, with the proviso that when R, is Si-(A) R is also Si-(A) Furtherin Chart B, B in Formula XVI is H OH,

and indicates attachment to the cyclopentane ring in alpha or betaposition.

The Chart B starting material VII is prepared as shown in Chart A. Thecompounds of Formula VIII in Chart A are included in Formula VII. Asdescribed above, 1518- PGE 15,8-PGE methyl ester, and IS S-PGE methylester, and fi-PGE, methyl ester acetate are obtained from Plexaurahomomalla (Esper), 1792, forma R. All of those compounds are encompassedby Formula VII, and thus, extnaction of this form of Plexaura homomallaprovides an alternative source of these starting materials.

Referring to Chart B, the starting material VII can be a mixture ofcompounds with regard to the configuration of C-11, or the startingmaterial can be stereochemically pure with respect to 0-11, dependingupon whether there has been an earlier separation of Hot and Hi? isomers(see above discussion of Chart A reactions).

The transformation of PGE-type starting material VII to PGF-type productXI involves reduction of a ring carbonyl to a ring hydroxy. This processis known in the art 14 for some of the compounds encompassed by FormulaVII, i.e., when CHART B COORI VII (hydrolysis or aleoholysls theconnguration at 0-11 is alpha and the configuration at C-15 is S.

For this carbonyl-to-hydroxy reduction, methods known in the art areused. See, for example, Pike et al., J. Org. Chem. 34, 3552 (1969). Useis made of any of the known ketonic carbonyl reducing agents which donot reduce ester or acid groups or carbon-carbon double bonds. EX-amples of those are the metal borohydrides, especially sodium,potassium, lithium, and zinc borohydrides, lithium (tri-tert-butoxy)aluminum hydride, metal trialkoxy borohydrides, e.g., sodiumtrimethoxyborohydride, and diisobutylaluminum hydride. The sodium,potassium, and zinc borohydrides are preferred for this reduction,especially zinc borohydride.

This carbonyl reduction usually produces a mixture of PGFa-type andPGFfl-type compounds, i.e., compounds with the alpha configuration andcompounds with the beta configuration for the hydroxy at 0-9. Thismixture of alpha and beta isomers is separated by methods known in theart, e.g., chromatography on silica gel. See Pike et al., ibid., forexample. If the Formula VII starting material is a mixture of and 11/3isomers, then this reduction will usually produce four isomers, i.e.,9a, 110:, 9a, 115, 9 3, 1100, and 95, 11/3. Those compounds are alsoseparated from such mixtures by silica gel chromatography.

Regarding the transformation of VII to IX in Chart B, it will beobserved that the parameters for VII are such that all VII compounds areincluded in IX. In other words, the transformation VII to IX is anoptional process step in proceeding from VII to X. The reason for thisis as follows. During the reduction of IX to X, the ratio of 9a-hydroxyand 9f3-hydroxy compounds formed will be different when R in IX ishydrogen than when R is Si-(A). For example, with the Formula IXcompound wherein R is hydrogen, G is and R O- represents HO---, i.e.,lla-hydroxy, sodium borohydride reduction gives 42 parts of thecorresponding Formula X 9a-hydroxy compound, and 58 parts of the 9B-hydroxy compound. But with the Formula IX compound wherein R ishydrogen, G is n osion,

and R O- represents (CH -Si-'O---, sodium borohydride reduction gives 85parts of the corresponding Formula X 9u-hydroxy compound and 15 parts ofthe 913- hydroxy compound. Similar differences are observed with theother isomers encompassed by Formula IX although not necessarily in thesame direction. Accordingly, whether R in Formula IX is to be hydrogenor -Si--(A) depends on the particular Formula X C-9 isomer desired andthe influence of silylation on the isomer ratio. For any particularFormula IX starting material, the latter is readily determined by smallscale reduction with and without silylation. When silylation beforecarbonyl reduction is indicated, largely for economic reasons, it ispreferred that A be methyl, i.e., that R be (CH Si.

With regard to the Formula X carbonyl reduction product (Chart B), whenthe method used to isolate said product does not remove any -Si(A)groups which may be present, that is accomplished as described above forthe removal of mSi(A)3 groups from Formula VII products (Chart A, VII toVIII). Also, when G in Formula X is H OCOCHI H oooorn the acetyl isremoved by alcoholysis also as described above for changing acetoxy atC-15 to hydroxy. These reactions are shown in Chart B as X to XI.

When R in Formula XI is methyl and the compound wherein R is hydrogen isdesired, that methyl ester is saponified, by methods known in the art.See, for example, Just et al., J. Am. Chem. Soc. 91, 5371 (1969). Thissaponification also changes a C-15 acetate to a C-lS hydroxy.

The compounds encompassed by Formula XI include the' known compounds PGFPGF and the methyl esters of those. Also included in Formula XVI are thecompounds 15fi-PGF ISB-PGF and the methyl esters of those.

When one of these Formula XI compounds has the R or epi configurationfor the hydroxy at Cl5, and the corresponding compound with the Sconfiguration at C-15 is desired, or when one of these Formula XIcompounds has the S configuration for the hydroxy at C-l5, and thecorresponding compound with the R or epi configuration at -15 isdesired, those desired compounds are made by the processes of Chart C.In Chart C, R R R B, and are as defined above.

The overall process scheme of Chart C is to start with one particular0-15 isomer of a compound encompassed by Formula XI, i.e., either 15 (S)or 15 (R). The C-15 hydroxy of that isomer is oxidized to a ketoniccarbonyl (XII). Then, after an optional silylation of the C-9 and C-11hydroxy groups (XIII), the C-15 carbonyl is reduced back to a secondaryhydroxy group. That reduction produces two C-15 hydroxy isomers, onewith S configuration and one with R or epi configuration. After re- 16moval of any silyl groups, the isomers XIV and XV are separated. One ofthe isomers will be the same compound used as starting material (XI).

CHART C COORl HO B X! l(oxldatlon) coon,

5 HO 0 x11 1 (silylation) T COOR:

2 R50 XIII 1 (reduction) 1 (hydrolysis) COORI XIV no H on xv The otherisomer will be the desired product. The starting material isomer isrecycled to produce more of the desired isomer. This reaction scheme haspreviously been used to transform PGF to 15;3-PGF See Pike et al., J.Org. Chem. 34, 3552 (1969').

Referring now to Chart C, any oxidizing agent can be used which willoxidize an allylic alcohol to an a,punsaturated ketone or aldehyde.Examples of those are 2,3- dichloro-5,6-dicyano-1,4 benzoquinone,activated manganese dioxide, or nickel peroxide (see Fieser et al.,Reagents for Organic Syntheses, John Wiley & Sons, Inc., New York, N.Y.,1967, pp. 215, 637, and 731). Alternatively, these oxidations arecarried out by oxygenation in the presence of the15-hydroxyprostaglandin dehydrogenase of swine lung (see Arkiv for Kemi25, 293 (1966). These reagents are used according to procedures known inthe art. See, for example, J. Biol. Chem. 239, 4097 (1964).

Regarding the transformation of XVII to XVIII in Chart C, thesesilylations are carried out as described above for the Chart A and ChartB silylation.

The carbonyl reduction of XIH to XIV is carried out as described abovefor the transformation of PGE-type Formula IX compounds to PGF-typeFormula X compounds. As for those reductions, the sodium, potassium andzinc borohydrides are preferred as reducing agents, especially zincborohydride.

When the method used to isolate the carbonyl reduction product does notremove any Si-(A) groups which may be present, that is accomplished asdescribed above for the removal of-Si-(A) groups from Formula VIIproducts (Chart A, VII to VIII).

The Formula X'IV and XV products are separated from each other bymethods known in the art, for example, silica gel chromatography. See,for example, Pike et al., J. Org. Chem. 34, 3552 (1969) for this type ofseparation.

If one of the isomers or isomer mixtures of Formulas XIV or XV is notdesired for a pharmacological use as such or transformed to esters orpharmacologically acceptable salts as described above, that isomer orisomer mixture is recycled as a Formula XI starting material in theprocesses of Chart C to produce additional of the desired isomer.

In Chart D is shown an optional route to PGF starting with 15}8-PGAacetate methyl ester, the most abundant component of Plexaura homomalla(Esper), 1792, forma R. In Chart E is shown a route to PGE starting withPGA readily obtained as described above from Plexaura homomalla (Esper),1792, forma S.

CHART D l-fl-P GAz acetate methyl ester (oxidation) 15fl-P GA: acetatemethyl ester or end B 10,11-epoxides (reduction) 15 9-1 GE; and116,15fi-P GEB 15-ecetete methyl esters (separation) 15fl-PGE215-acetate methyl ester (silylatlon) 155-? GE 11-Sl-(A) ether 15-aeetatemethyl ester (reduction, hydrolysis) 1519-1? GFz,l and 155-1 GFrp15-ecetate methyl esters (separation) 15t3-PGF1.l 15-aeetate methylester (saponlflcetion) (oxidation) 15-oxo-P GFaa (sllylatlon) 15-oxo-PGF 9,11-di-Sl-(A); ether (reduction, hydrolysis) lsfl'PGFhx and P GFaa(separation) t! l-BP GF-zn PGF a CHART E PGA,

(separation) PGE:

All of these Chart D and Chart E reactions and reagents for effectingthem are described generically and specifically above, and all areexemplified below in the preparations.

The invention is more fully understood by the following preparations andexamples:

All temperatures are in degrees centigrade.

Ultraviolet spectra are recorded on a Cary Model 15 spectrophotometer.

The collection of chromatographic eluate fractions starts when theeluant front reaches the bottom of the column.

Brine, herein, refers to an aqueous saturated sodium chloride solution.

The A-IX solvent system used in thin layer chromatography (TLC) is madeup from ethyl acetate-acetic acid- 2,2,4-trimethylpentane-Water (:20:50:according to M. Hamberg and B. Samuelsson, J. Biol. Chem. 241, 257(1966).

Preparation 1 To distinguish Plexaura homomalla (Esper), 1792, forma Rfrom Plexaura homoma lla (Esper), 1792, forma S, a TLC method is used. Aspecimen approximately 2 cm. in length is harvested and placed in asmall vial, with a small amount of water if necessary to insure it iswet, and kept closed for 6-24 hrs. About one ml. of methanol is thenadded and the sample is either shaken for 2 hrs. at about 25 C. or isstored for 16-24 hrs. at about 10 C. A sample of the liquid (10-21 A) isspotted on a TLC plate. It is preferred to use a fluorescent-treatedsilica gel plate, e.g., Uniplate Silica Gel GF (Analtech, Inc., Newark,Del.). As reference standards, spots of PGA and 15B-PGA are alsoapplied. The plate is developed in the A-IX system. The spots arefinally visualized with vanillin-phosphoric acid spray (McAleer, Arch.Biochem. E. Biophys. 66, (1957)). Comparison of the unknown with the tworeference spots is then made and the identity of the coral established(forma S corresponding ot PGA forma R to 15fi-PGA Preparation 2.PGA and5,6-trans-PGA Separation of PGA from 5,6-trans-PGA is done on achromatographic column using a silver-saturated ion-exchange resin.Preferably a macroreticular ion exchange resin is used, eg a sulfonatedstyrene-divinylbenzene copolymer having surface area of 40-50 sq. m./g.,30-40% porosity, and total exchange capacity of 4.5-5.0 meq. per gram ofdry resin, for example Amberlyst 15, available from Rohm and Haas Co.,Philadelphia, Pa. The acidform: resin is packed in a column, washed withwarm water, and converted to the silver form by passing a 10% silvernitrate solution through the column until the effluent shows a pH of3.5-4.0. The column is then washed with water to remove ionic silver,and finally with denatured ethanol (Type 3A). A solution of a mixture ofPGA; and 5,6-trans-PGA e.g. fractions 15-18 of EX- ample 7 below, inethanol is charged to the column. Elution with 3A alcohol then yieldsfractions which are combined according to their content of 5,6-trans-PGA(less polar) or PGA Testing for the presence of 5,6-trans- PGA or PGA inthe eluate is conveniently done by TLC using silver nitrate-treatedsilica gel plates (e.g. Analtech Uniplates dipped in saturated ethanolicsilver nitrate and dried) and developing with the A-IX system. R: of PGAis 0.45; R, of 5,6-trans-PGA is 0.50. Combined fractions areconcentrated, partitioned between dichloromethane and a little water,dried over sodium sulfate, and concentrated under reduced pressure toyield the title compounds.

For quantitatively assaying the 5,6-trans-PGA content of mixtures of PGAand 5,6-trans-PGA a combination thin-layer-spectrophotometric assay isused. Silica gelimpregnated glass microfiber sheet (e.g. ITLC sheets ofthe Gelman Instrument Co., Ann Arbor, Michigan) are impregnated withsilver nitrate, using 5% ethanolic silver nitrate and drying. Spots of100 to 200 [Lgof the PGA: mixture are applied and developed in thesolvent system 2,2,4-trimethylpentane :ethyl acetate: acetic acid water(100235 18:10, upper phase). The sheet is dried and sprayed withRhodarnine 6G (Applied Science Co., State College, Pa.) and viewed underultraviolet light. The areas containing the cis and trans materials (R;of PGA =O.6; R; of 5,6-trans-PGA =0.7) are marked, then excised andeluted with methanol (1.9 ml.) and potassium hydroxide solution (0.1 ml.of 45%). After incubation at 40 for 30 min., the respective solutionsare centrifuged and analyzed spectrophotometrically at 278 nm.

Following the procedure of Preparation 2, 5,6-transfl-PGA is separatedfrom 15/3-PGA Preparation 3.-PGA 15-acetate methyl ester, separationfrom 5,6-trans-PGA IS-acetate methyl ester A mixture of PGA 15-acetatemethyl ester and 5,6- trans-PGA l5-acetate methyl ester (11.0 g., 85:15)is dissolved in 415 ml. of a solution of methanol-wateracetic acid(95-5-04) and mercuric acetate (6.1 g.), and left standing at about 25C. for 30 min. Water (250 ml.) is added and the-mixture extracted twicewith 700 ml. of Skellysolve B. The Skellysolve B phase is washed with100 ml. of 60% methanol, dried over sodium sulfate, and concentrated toan oil (4.35 g.) having a high content of 5,6-trans-PGA l5-acetatemethyl ester. The aqueous methanol phase is acidified with 32 ml. of 6N. hydrochloric acid and the mixture is extracted with two portions of700 ml. of Skellysolve B. The organic phase is dried over sodium sulfateand concentrated to an oil (5.53 g.). This last meterial is subjected tothe same procedures again, using 350 ml. of the methanol-water-aceticacid and 4.6 g. of mercuric acetate. There is recovered from the work-upof the aqueous methanol phase a fraction (3.92 g.) of the title compoundcontaining only a small percentage of the 5,6-trans-PGA compound.

Following the procedure of preparation 3, 5,6-trans- 15/3-PGA 15-acetatemethyl ester is separated from 1513- PGA l5-acetate methyl ester.

'Preparation 4.15p-PGA methyl ester A solution of 70% aqueous perchloricacid (50 drops) in 50 drops of water is added to a solution of 15B-PGAacetate methyl ester (Example 3 below, 2.0 g.) in 100 ml. of methanol.The mixture is stirred for 15 hrs. at 25 C. and then diluted with 80 ml.of water. The methanol is removed under reduced pressure, and theaqueous residue is extracted with ethyl acetate. The extract is washedsuccessively with water and brine, dried with anhydrous sodium sulfate,and evaporated. The residue is chromatographed on 200 g. of silica gel,eluting with 2.5 l. of a gradient of -70% ethyl acetate in Skellysolve B(a mixture of isomeric hexanes), collecting 100-ml. fractions. Fractions15-19 are combined and evaporated to give 727 mg. of l5F-PGA methylester.

Preparation 5 .-PGA methyl ester A solution of ISfi-PGA methyl ester(Preparation 4, 250 mg.) in 20 ml. of anhydrous tetrahydrofuran iscooled to 0 C. in an atmosphere of nitrogen. Tributylamine (1.5 ml.) isadded, and the mixture is stirred at 0 C. While adding methanesulfonylchloride (1 ml.) dropwise. The mixture is stirred 30 minutes at 0 C.Then, 10 ml. of Water is added, and the mixture is allowed to warm to 25C. and is stirred for one hour. The tetrahydrofuran is evaporated underreduced pressure, and the aqueous residue is extracted with ethylacetate. The extract is washed successively with one N hydrochloricacid, water, and brine, dried with anhydrous sodium sulfate, andevaporated. The residue is chromatographed on 30 g. of silica gel,eluting with 800 ml. of a gradient of 2070% ethyl acetate in SkellysolveB, collecting 25 ml. fractions. Fractions 14-16 are combined andevaporated to give 58 mg. of PGA methyl ester. Fractions 12 and 13 arecombined to give 49 mg. of the starting material, IS/S-PGA methyl ester.

Following the procedure of Preparation 5, PGA methyl ester istransformed to a mixture of PGA and 15B- PGA methyl esters, the twocompounds being separated as in Preparation 5.

Preparation 6.-l5fl-PGA 10,11-epoxide acetate methyl ester Hydrogenperoxide (350 ml.; 30% aqueous) is added with stirring to a solution ofISB-PGA acetate methyl ester (Example 3 below, 265 g.) in 5000 ml. ofmethanol under a nitrogen atmosphere at 20 C. Then, one N aqueouspotassium hydroxide solution (50 ml.) is added gradually during one hourWth stirring at 20 C. The mixture is stirred an additional 2 hours at 20C. Then, one N hydrochloric acid ml.) is added, and the methanol isremoved under reduced pressure at 35 C. The residue is dissolved in 3000ml. of ethyl acetate, and the solution is washed 3 times with SOO-ml.portions of water. The combined water washes are extracted with 300 ml.of ethyl acetate. The two ethyl acetate solutions are combined, washedwith brine, dried with anhydrous sodium sulfate and evaporated to give2.75 g. of a mixture of the alpha and beta 10,11-epoxides of l5 3-PGAacetate methyl ester.

Preparation 7.1513-PGE 15-acetate methyl ester and 115,15B-PGEIS-acetate methyl ester Granular aluminum metal (50 g.) is added to asolution of mercuric chloride (50 g.) in 2 l. of water. The mixture isswirled until hydrogen gas evolution starts to become vigorous (about 30seconds). Then, most of the aqueous solution is decanted, and the restis removed by rapid filtration. The amalgamated aluminum is washedrapidly and successively with two 200-ml. portions of methanol and two200-ml. portions of anhydrous diethyl ether. The amalgamated aluminum isthen covered with anhydrous diethyl ether until used.

Methanol (250 m1.) and Water (25 ml.) are added to a solution of amixture of the alpha and beta 10,11- epoxides of 15fi-PGA acetate methylester (Preparation 6, 275 g.) in 2500 ml. of diethyl ether. The mixtureis cooled to 10 C. and the amalgamated aluminum prepared as above from50 g. of aluminum metal is added. The mixture is stirred and maintainedat about 25 C. with external cooling. After one hour, amalgamatedaluminum prepared as above from 50 g. of aluminum metal is added. Afteran additional hour of stirring at 25 C., amalgamated aluminum preparedas above from 50 g. of aluminum metal and also 25 ml. of water areadded. After an additional hour of stirring at 25 C., g. of magnesiumsulfate is added as a filter aid, and the mixture is filtered. Thefilter cake is washed thoroughly with dichloromethane, and the combinedfiltrate and washings are evaporated at 25 C. under reduced pressure togive 21 a mixture (247 g.) of 15B-PGE 15-acetate methyl ester andIIfiJSB-PGE 15-acetate methyl ester.

Part of this mixture (210 g.) is chromatographed on 30 kg. of silica gelWet-packed with 60 l. of 25% ethyl acetate in Skellysolve B (6-inchdiameter column), eluting successively with 60 1. portions of 25%, 30%,35%, 40%, 45%, 50%, 55%, and 60% ethyl acetate in Skellysolve B,collecting 4 1. fractions. Fractions 71-76 are combined and evaporatedto give 27 g. of 11B,15,8-PGE 15-acetate methyl ester. Fractions 81-98are combined and evaporated to give 115 g. of IS/S-PGE; l5-acetatemethyl ester.

Preparation 8.-l5/3-PGE 1l%i(CH ether 15-acetate methyl esterHexamethyldisilazane (100 g.) and then trimethylchlorosilane (20 g.) areadded to a solution of ISB-PGE 15-acetate methyl ester (Preparation 7)in 400 ml. of tetrahydrofuran with vigorous stirring at 25 C. undernitrogen. The reaction mixture is maintained in the range 20 to 25 C. byexternal cooling, and is stirred 2 hours under nitrogen. Then, themixture is evaporated at 50 C. at reduced pressure. The residue is mixedwith 150 ml. of toluene, and the mixture is filtered through a pad ofdiatomaceous earth. The filtrate is evaporated at 50 C. under reducedpressure. The residue is mixed with 150 ml. of toluene, and again thetoluene is removed under reduced pressure at 50 C. to give 75 g. of15/3- PGE l1-Si-(CH ether IS-acetate methyl ester.

Following the procedure of Preparation 8, 1118,15 PGE 15-acetate methylester is transformed to the corresponding 11Si-(CH ether.

Also following the procedure of Preparation 8, but using larger amountsof hexamethyldisilazane and trimethylchlorosilane, l5f3-PGE methylester, 15B-PGE 115,15fi-PGE methyl ester, and 115,155-PGE aretransformed to the corresponding 11,15-di-Si-(CH ethers.

Preparation 9.-15;8PGF IS-acetate methyl ester and ISB-PGF IS-acetatemethyl ester Sodium borohydride (1.42 g.) is added in one portion to asolution of ISB-PGE 11-Si-(CH ether IS-acetate methyl ester (Preparation8, 30.7 g.) in 500 ml. of absolute ethanol at C. with stirring. Themixture is stirred at 0 C. for 3.5 hours. Then, 10 ml. of glacial aceticacid is added slowly with stirring at 0 C. Then, 100 ml. of water isadded, and the mixture is allowed to warm to 25 C. with stirring, and isstirred 15 hours at 25 C. The ethanol is evaporated under reducedpressure, and the residue is mixed with 400 ml. of brine. The mixture isextracted with 3 portions of ethyl acetate (400 ml., 250 ml., and 150ml.). The combined extracts are washed successively with two 100-ml.portions of water, 100 ml. of saturated aqueous sodium bicarbonatesolution, two 100-ml. portions of brine, dried with anhydrous sodiumsulfate, and evaporated under reduced pressure to give 24.5 g. of amixture of ISB-PGF IS-acetate methyl ester and lSfl-PGF IS-acetatemethyl ester.

Preparation l0.-15,B-PGF and 15 3-PGF Aqueous sodium hydroxide solution(10%; 275 ml.) is added to a solution of 48 g. of a mixture of ISB-PGFIS-acetate methyl ester and 15;BPGF IS-acetate methyl ester (Preparation9) in 350 ml. of methanol at 0 C. with stirring under nitrogen. Themixture is allowed to warmt'cl 25 C. with stirring, and is stirred 3hours at 25 C. Then, the methanol is evaporated under reduced pressureat 35 C. The aqueous residue is cooled and extracted once with a mixtureof diethyl ether and dichloromethane (1:1). Then, the aqueous residue isacidified with 260 ml. of 3 N hydrochloric acid, saturated with sodiumchloride, and extracted with 3 portions of ethyl acetate (400 ml., 250ml., and 150 ml.). The combined extracts are washed successively withtwo 100-ml. portions of water and two 100-ml. portions of brine, driedwith anhydrous sodium sulfate, and evaporated to give 42 g. of a mixtureof ISB-PGF and 15fl-PGF Preparation 1l.-15-oxo-PGF and 15-oxo-PGF Themixture of lSfi-PGF and 15 3-PGF (42 g.) obtained as in Preparation 10is dissolved in 950 ml. of dioxane. To this solution at 25 C. is added2,3-dichloro- 5,6-dicyano-1,4-benzoquinone (40 g.). This mixture isstirred 18 hours at 50 C. under nitrogen. The mixture is then cooled teo25 C. and filtered. The filter cake is washed with dichloromethane, andthe combined filtrate and washing are evaporated under reduced pressureat 45 C. to give a mixture (66 g.) of -oxo-PGF and 15-oxo-PGF Part ofthis mixture (33 g.) is chromatographed on 3 kg. of acid-washed silicagel, eluting successively with 10 l. 60%, 101. 70%, 1'01. 80%, 201. 90%ethyl acetate in Skellysolve B, 15 l. ethyl acetate, and 10 l. 5%methanol in ethyl acetate, collecting 650 ml. fractions. Fractions 4253are combined and evaporated to give 8.3 g. of 15-oxo-PGF Fractions 64-85are combined and evaporated to give 3.3 g. of 15-oxo-PGF Preparationl2.-PGF and IS S-P61 Hexamethyldisilazane (70 ml.) andtrimethylchlorosilane (14 ml.) are added with vigorous stirring to asolution of 15-oxo-PGF (Preparation 11, 3.0 g.) in 350 ml. oftetrahydrofuran at 25 C. under nitrogen. The mixture is stirred 18 hoursat 25 C. under nitrogen. Then, the mixture is evaporated under reducedpressure at 50 C. Toluene (100 ml.) is added to the residue, and themixture is filtered through a pad of diatomaceous earth. The filtrate isevaporated, and 100 ml. of toluene is added to the residue. This mixtureis evaporated under reduced pressure to give the 9,11-di-Si-(CH ether of15-oxo- PGF This disilyl ether is dissolved in 20 ml. of1,2-dimethoxyethane. Sodium borohydride (680 mg.) is suspended in 65 ml.of 1,2-dimethoxyethane at 0 C. under nitrogen. Anhydrous zinc chloride(1.23 g.) is added to this suspension, and the mixture is stirred 30minutes at 0 C. Then, the solution of the disilyl ether is addeddropwise during 10 minutes with stirring at 0 C. The resulting mixtureis allowed to warm to C. with stirring, and is stirred 4 hours at 25 C.Then, ml. of water is added, followed by 8 ml. of glacial acetic acid.This mixture is stirred 15 hours at 25 C. The mixture is then pouredinto a mixture of ice and 100 ml. of 0.5 N hydrochloric acid. Thatmixture is saturated with sodium chloride, and then extracted withseveral portions of ethyl acetate. The combined extracts are washed withbrine, dried with anhydrous sodium sulfate, and evaporated under reducedpressure. The residue (3.2 g.) is chromatographed on 600 g. ofacid-washed silica gel, eluting successively with 5 l. of 75% ethylacetate in Skellysolve B, 5 l. of 90% ethyl acetate in Skellysolve B,and 5 l. of a gradient of 90% ethyl acetate and 10% methanol in ethylacetate, collecting 550 m1. fractions. Fractions 21-26 are combined andevaporated to give 543 mg. of 15/9-PGF Fractions 28-36 are combined andevaporated to give 1.62 g. of PGF Preparation 13.15-oxo-PGF Followingthe procedure of Preparation 11, ISB-PGF- is oxidized to 15-oxo-PGFPreparation 14.PGF and ISfl-PGF- Preparation l5.PGE and llfi-PGEHexamethyldisilizane (1 ml.) and trimethylchloro 75 silane (0.2 ml.) areadded with stirring to a solution of PGA;, (250 mg.) in 4 ml. oftetrahydrofuran at 0 C.

under nitrogen. This mixture is maintained at C. for 15 hours. Themixture is then evaporated under reduced pressure. Toluene is added andevaporated twice. Then the residue is dissolved in 6 ml. of methanol,and the solution is cooled to 20" C. Hydrogen peroxide (0.45 ml.; 30%aqueous) is added. Then, one N sodium hydroxide solution (0.9 ml.) isadded dropwise with stirring at 20 C. After 2 hours at 20 C., anadditional 0.3 ml. of the sodium hydroxide solution is added withstirring at P20 C. After another hour in the range to i20 C., anadditional 0.1 ml. of the sodium hydroxide solution is added. Then, 1.5ml. of one N hydrochloric acid is added, and the mixture is evaporatedunder reduce pressure. The resiue is extracted with ethyl acetate, andthe extract is washed successively with one N hydrochloric acid andbrine, dried with anhydrous sodium sulfate and evaporated. The residueis dissolved in 5 ml. of diethyl ether. To this solution is added 0.5ml. of methanol and 0.1 ml. of water. Then, amalgamated aluminum madefrom 0.5 g. of aluminum metal as described in Preparation 7 is added insmall portions during 3 hrs. at 25 C. Then, ethyl acetate and 3 Nhydrochloric acid are added, and the ethyl acetate layer is separatedand washed successively with one N hydrochloric acid and brine, driedwith anhydrous sodium sulfate, and evaporated. The residue ischromatographed on 50 g. of acid-washed silica gel, eluting first with400 ml. of a gradient of 50100% ethyl acetate in Skellysolve B, and thenwith 100 ml. of 5% methanol in ethyl acetate, collecting 25 ml.fractions. Fractions 9 and 10 are combined and evaporated to give 18 mg.of ll S-PGE Fractions 17-25 are combined and evaporated to give 39 mg.of PGE Preparation l6.PGE- (Refer to Chart C) (a) Silylation.-A mixtureof PGA (0.68 g.), 4 ml. of tetrahydrofuran (THF), and 1 ml. oftrimethylchlorosilane solution (5% in hexamethyldisilazane) is stirredunder nitrogen for 2 hrs. at about 25 C. Then the silylated material isconcentrated by removal of THF under reduced pressure, utilizing addedbenzene (10 ml.) to facilitate removal of THF.

(b) Oxidation-A cold (-40 C.) solution of the above silylated materialin ml. of isopropyl alcohol is mixed with 1.2 ml. of 30% aqueoushydrogen peroxide, followed by 1.5 ml. of 3 N. aqueous lithium hydroxideadded dropwise. The temperature is allowed to warm to about 30 C. Thereaction is continued until the PGA has been exhausted as shown by theabsence of PGA in a thin layer chromatographic (TLC) spot test using theA-IX system (Hamberg and Samuelsson, J. Biol. Chem. 241, 257 (1966). At30 C., the reaction time is about 3-4 hrs. After completion, 5 ml. of 1N. hydrochloric acid is added and the mixture is concentrated underreduced pressure. The residue is extracted with ethyl acetate, washedwith 0.5 N. hydrochloric acid and then brine, dried over sodium sulfate,and concentrated to the epoxide.

(c) Reduction and hydrolysis.A solution of the above epoxide in 20 ml.of THF and 2 ml. of methanol is stirred with 4 ml. of saturated aqueoussodium bicarbonate solution and cooled to 15 C. To the mixture is added,in portions with vigorous stirring, an aluminum amalgam made from 1 g.of powdered aluminum (Preparation 7). After stirring at about C. for 45min., a sample is analyzed by TLC for PGE and epoxide. Reaction iscontinued if necessary. When the epoxide is no longer present, thesupernatant suspension is decanted from the aluminum which is furtherwashed with ethyl acetate. The combined decantate and washes areconcentrated under reduced pressure. The residue is taken up in about15-20 ml. of ethyl acetate and shaken with 20 ml. of 1 N. hydrochloricacid. The layers are separated, the organic phase is washed with 24 0.5N. hydrochloric acid, and then brine, dried and concentrated to an oilyresidue of 0.837 g.

(d) Separation-A solution of the above residue in a small amount of 20%ethyl acetate-Skellysolve B (isomeric hexanes) is applied to achromatographic column of 65 g. of acid-washed silica, e.g. MallinckrodtSilicar CC-4. Elution with a gradient of 20-100% ethyl acetate-Skellysol've B gives fractions. Those fractions which are shown by TLCto contain the desired compound are combined, and concentrated. There isobtained in separate fractions PGE 0.5 g., and 11B-PGE 0.05 g.

Alternatively, the oily product from 0 above is triturated in ethylacetate-cyclohexane (1:1), cooled to about 10 C. and seededto yieldcrystalline PGE about 0.4 g. The mother liquor is subjected to silicagel chromatography to yield separate fractions of about 0.1 g. PGE and0.05 g. llp-PGE EXAMPLE 1 PGA PGA methyl ester, and PGA,, acetate methylester from Plexaura homomalla (Esper), 1792, forma S Colonies ofPlexaurw homomalla (Esper), 1972, forma S, collected from reefs off thenorth shore of Jamaica, are chopped into pieces several cm. in lengthfrozen. The pieces (500 g.) are then covered with methanol and themixture is maintained for 3 hours at 25 C. The mixture is then ground ina Waring blender and filtered, and the filtrate is evaporated underreduced pressure. The residue is dissolved in ethyl acetate, and thesolution is washed successively with one N hydrochloric acid, water, andsaturated aqueous sodium chloride solution, dried with anhydrous sodiumsulfate, and evaporated under reduced pressure. The oily residue ischromatographed on 2 kg. of acid-washed silica gel wet-packed withSkellysolve B (a mixture of isomeric hexanes), eluting with 24 l. of a25 to ethyl acetate in Skellysolve B gradient. The fractions whichcontain PGA acetate methyl ester, PGA methyl ester, and PGA as shown byTLC with the A-IX system are separately combined and evaporated to givethose three compounds, respectively, in the ratio 60:20:20.

EXAMPLE 2 ISfl-PGA compounds from Plexaura homomalla (Esper), 1792,forma R Following the procedures of Example 1, but replacing Plexaurahomomalla (Esper), 1792, forma S with Plexaura homomallw (Esper), 1792,forma R collected from reefs off the southeast shore of Florida nearMiami, there are obtained 15fi-PGA acetate methyl ester, 15,8- PGAacetate, 15B-PGA methyl ester, and lS S-PGA Likewise following theprocedures of Examples 1 and 2 but replacing methanol with acetone,there are obtained the respective PGA and 15fi-PGA compounds.

EXAMPLE 3 15p-PGA compounds from Plexaura homomalla (Esper), 1792, formaR Colonies of Plexaura homomalla (Esper), 1792, forma R, collected fromreefs off the southeast shore of Florida near Miami, are chopped intochunks several inches long. The chunks are frozen by contact with solidcarbon dioxide within one hour after removal from the reef waters. Thefrozen colony pieces are maintained in insulated boxes containing solidcarbon dioxide (temperature below about 20 C.) until the time forextraction. Then, the frozen colony pieces are ground to a smallparticle size (Mitts and Merrill hogger; average largest dimension about5 mm.). The particles (1500 g.) are then stirred at high speed with 5gallons of dichloromethane for 20 minutes at about 25 C. externaltemperature. The mixture of dichloromethane and particles is thenfiltered through a pad of diatomaceous earth, and the filtrate isevaporated to about a 2-liter colume at 30 C. under reduced pressure.The liquid which remains is washed with water, dried with sodiumsulfate, and evaporated at 30 C. under reduced pressure.

The oily residue (60 g.) is chromatographed on 3 kg. of silica gel wetpacked in Skellysolve B (a mixture of isomeric hexanes), elutingsuccessively with a gradient of 4 l. of Skellysolve B and 4 l. of 20%ethyl acetate in Skellysolve B, 27 l. of 20%, 18 l. of 50%, and 8 l. of75% ethyl acetate in Skellysolve B, collecting 600 ml. fractions.Fractions 39-60 are combined and evaporated to give 24.3 g. of ISp-PGA,acetate methyl ester. Between fractions 60 and 74 those fractions shownby TLC to contain fl-PGA acetate are combined and evaporated to yieldthat compound. Fractions 74-76 are combined and evaporated to give 1.03g. of 15,9-PGA methyl ester. Fractions 83-91 are combined and evaporatedto give 1.08 g. of 15fl-PGE 15-acetate methyl ester. Still laterfractions shown by TLC to contain 15 8-PGE methyl ester are combined andevaporated to yield that compound.

Detection of the respective compounds by TLC is done by methods known inthe art, e.g. by spotting the extract fractions on a TLC silica gelplate alongside spots of the authentic compounds, developing the platewith the A-IX system, and observing which spots of the extract fractionscorrespond exactly to the spots of the authentic compounds.

Following the procedures of Example 3, but substituting Plexaurahomomalla (Esper), 1792, forma S for the Plexaura homomalla (Esper),1792, forma R of that example, there are obtained the correspondingcompounds of 15 (S) configuration, viz.: PGA acetate methyl ester, PGAacetate, PGA methyl ester, PGE IS-acetate methyl ester, and PGE methylester.

EXAMPLE 4 Following the procedures of Example 3, but replacing Plexaurahomomalla (Esper), 1792, forma R, with Plexaura homomalla (Esper), 1792,forma S collected from reefs off the north shore of Jamaica, andsubstituting benzene for dichloromethane, there are obtained PGA acetatemethyl ester, PGA acetate, PGA methyl ester, PGE IS-acetate methylester, and PGE methyl ester.

EXAMPLE 5 PGA from Plexaura homomalla (Esper), 1792, forma S Colonies ofPlexaura homomalla (Esper), 1792, forma S, collected from reefs off thenorth shore of Jamaica, are chopped into pieces weighing less than about2 grams. The chopped material, wet with water, is stored in closedcontainers for about 24 hrs. at 25 C. or until PGA 14-acetate compoundsare no longer present, as shown by TLC analysis. The TLC sample is takenfrom the liquid phase of a mixture of a small representative sample ofthe coral, with adhering liquid, stirred or shaken in at least an equalvolume of acetone.

About one kg. of the water-wet chopped Plexaura homomalla is thenstirred in one 1. of acetone for about one hr. and filtered. The solidsare extracted in this manner four more times with acetone and finallyonce with dichloromethane. The aqueous acetone leach liquors areconcentrated under vacuum to about one-tenth their original volume. Tocontrol foaming, an antifoam agent is added, e.g. a polypropylene glycolsuch as Ucon LB-625 (Union Carbide), at a level of about one gram perkg. of wet colony pieces. The concentrate is then extracted three timeswith dichloromethane and the combined dichloromethane extracts areconcentrated to about one-fifth their original volume.

The dichloromethane concentrate is extracted seven times with an aqueoussolution of 0.5 M disodium orthophosphate, using 1.5 times the volume ofthe organic phase. To control emulsions, a 1% solution of antifoamagent, e.g. Ucon LB-625, in methanol is added, up to onetenth the volumeof the aqueous phase. The combined aqueous extracts are then acidifiedto pH 2-3 with 4 N 26 hydrochloric acid and extracted withdichloromethane. The dichloromethane extracts are decolorized withactivated carbon, dried over anhydrous sodium sulfate, and concentratedunder vacuum.

The residue is chromatographed on 2 kg. of acid-washed silica gelWet-packed with Skellysolve B, eluting with 25-100% ethyl acetate inSkellysolve B gradient. The fractions which contain PGA methyl ester andPGA as shown by TLC with the A-IX system are separately combined andevaporated to give those compounds, PGA being the principal product.

Following the procedures of Example 5, but subjecting the water-wetchopped Plexaura homomalla form S to a freezing step with solid carbondioxide after storage at 25 C. for 24 hrs., the frozen solids are storedbelow about 20 C. for a week. Then, they are thawed, stirred intoacetone, and thereafter processed to the same products as above.

EXAMPLE 6 15,8-PGA from Plexaura homomalla (Esper), 1792, forma RFollowing the procedures of Example 5 but replacing the Plexaurahomomalla (Esper), 1792, forma S of that example with chopped colonypieces of Plexaura homomalla (Esper), 1792, forma R, there are obtained15,3- PGA; methyl ester and 15 8-PGA methyl ester and 1513- PGA IS/B-PGAis the principal product.

EXAMPLE 7 PGA: from Plexaura homomalla (Esper), 1792, forma S.

Colonies of Plexaum homomalla (Esper), 1792, forma S, collected fromreefs off the north shore of Jamaica, are frozen by contact with solidcarbon dioxide within one hour after removal from the reef waters. Thefrozen colonies are maintained in insulated boxes containing solidcarbon dioxide (temperature below about 20 C.) until ready for thawing.Then, the frozen colonies (700 g.) are ground to a small particle size(Waring blender) and mixed with 1500 m1. of water. The mixture ismaintained about 20 hours. at about 25 C. with stirring. Then, the themixture is filtered through a pad of diatomaceous earth, and thefiltrate is acidified with concentrated hydrochloric acid to pH about2-3. The acidified filtrate is extracted four times with ethyl acetate.The extracts are combined, filtered, washed with brine, dried withanhydrous sodium sulfate, and evaporated under reduced pressure to give11 g. of oily residue.

The solid residue on the diatomaceous earth filter pad is stirred 2hours in methanol (enough to cover said residue) at 25 C. The mixture isthen filtered, and the filtrate is evaporated to give 14 g. of oilyresidue.

The oily residues are combined and chromatographed on 1500 g. ofacid-washed silica gel, eluting successfully with 8 l. of a 25 to 65%gradient of ethyl acetate in Skellysolve B, 8 l. of a 65 to gradient ofethyl acetate in Skellysolve B, and 5 l. of 2% methanol in ethylacetate, collecting 500 ml. fractions. (Skellysolve B is a mixture ofisomeric hexanes). Fractions 8-12 are combined and evaporated to give asmall amount of PGA containing a trace of PGA methyl ester. Fractions15-18 are combined and evaporated to give 9.54 g. of PGA Fractions 35-40are combined to give 0.414 g. of PGB EXAMPLE 8 15fl-PGA from PlexaumHomomalla (Esper), 1792, forma R.

Colonies of Plexaura homomalla (Esper), 1792, forma R, collected fromreefs oil the southeast shore of Florida near Miami, are chopped intochunks several inches long. The chunks are frozen by contact with solidcarbon dioxide within one hour after removal from the reef Waters. Thefrozen colony pieces are maintained in insulated boxes containing solidcarbon dioxide (temperature below about -20 C.) until ready for thawing.Then, colony pieces (600 g.) are mixed with 1500 ml. of water. Themixture is stirred and maintained at 25 C. for 23 hours. The mixture isthen filtered through a pad of diatomaceous earth, and the filtrate isacidified to pH about 2-3 with concentrated hydrochloric acid. Theacidified filtrate is extracted four times with ethyl acetate. Theextracts are combined, filtered, washed with brine, dried with anhydroussodium sulfate, and evaporated to give 9.2 g. of oily residue.

The solid residue on the diatomaceous earth pad is stirred 15 hours inmethanol (enough to cover said residue) at 25 C. The mixture is thenfiltered, and filtrate is evaporated. The residue is dissolved in ethylacetate, and the solution washed successively with 3 N hydrochloric acidand brine, dried with anhydrous sodium sulfate, and evaporated to give5.83 g. of an oily residue.

The second oily residue and 8.2 g. of the first oily residue arecombined and chromatographed on one kg. of acid-washed silica gel,eluting successively with 3-1. Portions of 25%, 35%, 45%, 55%, and 65%ethyl acetate in Skellysolve B, collecting 500-ml. fractions. Fractions18-22 are combined and evaporated to give 5.54 g. of 15fl-PGA Fractions15-17 are combined and evaporated to give 1.37 g. of 15,3-PGA methylester.

We claim:

1. In a process for extracting prostanoic acid derivatives from coloniesor colony pieces of the marine invertebrate Plexaura homomalla (Esper),1792, forma S or the marine invertebrate Plexaura homomalla (Esper),1792, forma R, the improvement wherein said colonies or colony pieces,prior to extraction, are maintained in contract with water in atemperature range up to 50 C. until substantially all of the C-l5acetates of the prostanoic acid derivatives originally present in thecolonies or colony pieces are transformed to C- hydroxy prostanoic acidderivatives.

2. A process improvement according to claim 1 wherein the colonies orcolony pieces are cooled to a temperature at least as low as +5 C. priorto maintaining in contact with water.

3. A process improvement according to claim 1 wherein the colonies orcolony pieces are frozen and thawed prior to maintaining in contact Withwater.

4. A process improvement according to claim 1 wherein after the coloniesor colony pieces are maintained in contact with water untilsubstantially all of the C-15 acetates of the prostanoic acidderivatives originally present in the colonies or colony pieces aretransformed to C-15 hydroxy prostanoic acid derivatives, they are cooledto a temperature at least as low as +5 C. prior to extraction.

5. A process improvement according to claim 1 wherein after the coloniesor colony pieces are maintained in con- 28 tact with water untilsubstantially all of the C-15 acetates of the prostanoic acidderivatives originally present in the colonies or colony pieces aretransformed to C-15 hydroxy prostanoic acid derivatives, said coloniesor colony pieces are frozen and thawed prior to extraction.

6. A process improvement according to claim 1 wherein the marineinvertebrate is Plexaura homoma'lla (Esper), 1792, forma S.

7. A process improvement according to claim 6 wherein the C-15 hydroxyprostanoic acid derivatives are PGA and PGA methyl ester.

8. A process improvement according to claim 1 wherein the marineinvertebrate is Plexaura homomalla (Esper), 1792, forma'R.

9. A process improvement according to claim 8 wherein the- C-15 hydroxyprostanoic acid derivatives are 15;;- PGA and 15fi-PGA methyl ester.

10. A process improvement according to claim 1 wherein said temperaturerange is 20 to 40 C.

11. A process improvement according to claim 1 wherein the quantity ofwater contacting the colonies or colony pieces is at least equal inweight to the dry solids content of the colonies or colony pieces.

12. A process improvement according to claim 1 wherein the colonies orcolony pieces of the marine invertebrate are maintained in contact withwater in a temperature range of about 20 to 40 C. until substantiallyall of the C-15 acetates of the prostanoic acid derivatives originallypresent in the colonies or colony pieces are transformed to C-l5 hydroxyprostanoic acid derivatives, said water being at least equal in weightto the dry solids content of the colonies or colony pieces, thereafterthe colonies or colony pieces being frozen to and maintained in thetemperature range of about 20 to C. until thawed.

13. A process improvement according to claim 12 wherein the marineinvertebrate is Plexaura homomalla (Esper), 1792, forma S.

14. A process improvement according to claim 12 wherein the marineinvertebrate is Plexaura homomalla (Esper), 1792, forma R.

References Cited Weinheimer et al., Tet Letters S (1969).

LORRAINE A. WEINBERGER, Primary Examiner R. GERSTL, Assistant ExaminerUS. 01. X.R. 260348 A, 448.8 RR, 488 R, 514 D; 424-305, 311, 317

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTIONPATENT NO. 5,794,675 DATED February 26, 1974 INVENTOR(S) Ramon D.Hamilton et al.

It is certified that error appears in the above-identified patent andthat said Letters Patent are hereby corrected as shown below: Column 5,lines 56-37, "l-5BPGA should read l5B-PGA Column 5, line 65, "theacetate ester" should read acetate methyl ester Column 15, l l ne 6,should read O OH H OH Column 17, lines 51-52, "l-B-PGA acetate methylester" should read 15B-PGA acetate methyl ester line 75,"lBPGF shouldread 15B-PGF2 Column 25, line l l, "reslue" should read residue Column 2l, line 24, "in length frozen" should read l n length and frozen Column27, l l ne 51, "contract" should read contact Signed and Sealed thisTwenty-seventh Day Of July 1976 [SEAL] Attest:

RUTH C. MASON C. MARSHALL DANN Arresting Officer Commissioner of Parentsand Trademarks

